This invention relates to a method for assaying protein C (hereinafter abbreviated as PC) and to a kit for measuring PC activity. More particularly, it relates to an enzymatic method for directly measuring the activity of PC without isolating it from the plasma, and to a measuring kit for the same.
PC is a vitamin-K-dependent plasma protein, which can be converted to activated protein C (hereinafter abbreviated as PCa) by the action of thrombin-thrombomodulin complex in the presence of Ca.sup.2+ ions. PCa is receiving attention as a plasma protein that is capable of selectively degrading blood coagulation factors V and VIII, thus exhibiting a powerful anti-coagulant action, and of liberating vascular plasminogen activator, thereby accelerating fibrinolysis.
It is known that PC level in the blood decreases as a result of disseminated intravascular coagulation syndrome (DIC) and hepatic diseases, such as hepatocirrhosis and chronic hepatitis. Recently a new PC deficiency disease accompanied by multiple thrombosis was reported. In addition, the effect of PC content upon the efficacy of blood coagulation factor (e.g., factor IX) preparations has been a subject of major concern.
Under the circumstances, there has been a demand for a simple method for correctly assaying PC in the plasma to diagnose the aforementioned PC deficiency disease, DIC and hepatic diseases, and to check the efficacy of various blood coagulation factor preparations.
Several methods are now used for quantitative analysis of PC, including the enzymatic method which employs a synthetic peptide substrate specific to PCa. But direct measurement of the activity of PC in the plasma by this type of method has been difficult, because the blood contains PC inhibitors and other interfering substances which, during activity measurement, act upon the synthetic peptide substrate without being inhibited by antithrombin III.
It is therefore necessary to previously isolate PC from the plasma by means of an antibody column or an adsorbent. But such a pretreatment, which requires a considerable amount of sample blood and needs much time and labor, is unsuitable for rapid treatment of a vast number of samples. In addition, the use of an adsorbent suffers from the low recovery rate of PC, while the use of an antibody column is very costly, although the PC recovery rate is satisfactory.
A biological method utilizing the anticoagulating action of PC is also known, in which PC, after being activated with snake venom, is added to a blood coagulation system and its action to prolong the coagulation time is measured. But the sensitivity of this method is not so high.
Immunological methods are also known, in which the amount of PC antigen is measured through an antigen-antibody reaction using a polyclonal or monoclonal antibody (the radioimmunoassay, enzyme immunoassay and Laurell's method). The techniques of this type are capable of correctly measuring the amount of PC antigen, but the disadvantages are that the antibody used is very costly, a long time is needed for measurement, and it is impossible to judge whether the PC tested has normal activity or not.
As a result of intensive studies to establish a new analytical method for PC free of the above-mentioned problems, we have succeeded in accomplishing this invention.